Conjugates of sodium channel blockers and methods of using the same

ABSTRACT

Compounds of the general formula P 1 -L-P 2 ; wherein “P 1 ” is a pyrazinoylguanidine sodium channel blocker, “L” is a linking group, and “P 2 ” is either (i) a pyrazinoylguanidine sodium channel blocker or (ii) a P2Y 2  receptor agonist, are disclosed. Pharmaceutical formulations containing the same and methods of use thereof to hydrate mucosal surfaces such as airway mucosal surfaces are also disclosed.

RELATED APPLICATIONS

[0001] This application is a divisional of co-pending U.S. patentapplication Ser. No. 09/628,978, filed Jul. 19, 2000, which claims thebenefit of U.S. Provisional Application No. 60/144,479, filed Jul. 19,1999, the disclosure of which is incorporated herein by reference in itsentirety.

STATEMENT OF FEDERAL SUPPORT

[0002] This invention was made with Government support under Grant No.HL51818 from the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to conjugates of sodium channelblockers, and particularly covalent conjugates comprising apyrazinoylguanidine sodium channel blocker and another compound such asanother pyrazinoylguanidine sodium channel blocker or a P2Y₂ receptoragonist.

BACKGROUND OF THE INVENTION

[0004] U.S. Pat. No. 4,501,729 to Boucher describes the use ofrespirable or non-respirable amiloride to hydrate airway mucoussecretions, and U.S. Pat. No. 5,656,256 to Boucher and Stutts describesthe use of respirable or non-respirable benzamil and phenamil to hydratelung mucus secretions. U.S. Pat. No. 5,789,391 to Jacobus describesmethods of treating sinusitis by administering uridine triphosphates(UTP) and related compounds such as P₁,P⁴-di(uridine-5′ tetraphosphate(U₂P₄) in order to promote drainage of congested fluid in the sinuses.

[0005] U.S. Pat. No. 5,292,498 to Boucher describes nucleotides,particularly P2Y₂ receptor agonists, that can be used to hydrate airwaymucus secretions. Dinucleotides that can be used to hydrate airway mucussecretions are described in U.S. Pat. No. 5,635,160 to Stutts et al..Additional compounds that are P2Y₂ receptor ligands and can be used tohydrate airway mucus secretions are disclosed in W. Pendergast et al.,U.S. Pat. No. 5,837,861, along with U.S. Pat. Nos. 5,763,447 to Jacobusand Leighton, and 5,789,391 to Jacobus et al.

SUMMARY OF THE INVENTION

[0006] A first aspect of the present invention is a compound(hereinafter also referred to as an “active compound” or “active agent”)of the general formula P₁-L-P₂; wherein “P₁” is a pyrazinoylguanidinesodium channel blocker, “L” is a linking group, and “P₂” is either (i) apyrazinoylguanidine sodium channel blocker or (ii) a P2Y₂ receptoragonist. An advantage of compounds of the present invention is that theyare substantially non-absorbable, or absorption-retardant or exhibitdelayed absorption on mucosal (e.g., airway, gastrointestinal) surfaces,thereby contributing to a prolonged mode of action and fewer systemicside effects.

[0007] A second aspect of the present invention is a compositioncomprising an active compound as defined above in an effectivetherapeutic amount, in a pharmaceutically acceptable carrier.

[0008] A third aspect of the present invention is a method of treating amucosal surface in a subject in need thereof, comprising administeringan active compound as described herein in an amount effective to treatthe subject. In general, treatment of the subject will mean that themucosal surface being treated with a compound or composition of thepresent invention will be hydrated, or that the compound or compositionbeing used will block or otherwise retard the absorption of liquid ontoor onto the mucosal surface, or that the mucosal surface will otherwiseexhibit an increased volume of liquid on the mucosal surface.

[0009] A fourth aspect of the present invention is the use of an activecompound as described above for the preparation of a medicament fortreating a mucosal surface in a subject in need thereof, as describedherein.

[0010] The foregoing and other objects and aspects of the presentinvention are explained in detail in the specification set forth below.

BRIEF DESCRIPTION OF DRAWINGS

[0011]FIG. 1 is a graphical illustration of the effect of apical washoutof a compound of the present invention, where wash-out correlates withcellular uptake. A range of reversibilities is shown, with the compoundCF-519 being completely reversible.

[0012]FIG. 2 is an example of a confocal microscopy assay of drug uptakeinto cultured airway epithelia. In this assay, a compound (10⁻⁴ M) isplaced on the airway surface and fluorescence from the cells collectedby x-z scanning confocal microscopy. The images shown on the left depictfluorescence in the cells 20 minutes after exposure to amiloride,benzamil and phenamil. Quantitation of the drug uptake is graphicallyillustrated on the right in terms of units of fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will now be described more fullyhereinafter with reference to the accompanying figures, which furtherillustrate the invention described herein. This invention may, however,be embodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

[0014] The terminology used in the description of the invention hereinis for the purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, iunless thecontext clearly indicates otherwise.

[0015] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety.

[0016] The term “alkyl” or “loweralkyl” as used herein refers to C1 toC4, C6 or C8 alkyl, which may be linear or branched and saturated orunsaturated. Cycloalkyl is specified as such herein, and is typicallyC3, C4 or C5 to C6 or C8 cycloalkyl. Alkenyl or loweralkenyl as usedherein likewise refers to C1 to C4 alkenyl, and alkoxy or loweralkoxy asused herein likewise refers to C1 to C4 alkoxy. The term “aryl” as usedherein refers to C3 to C10 cyclic aromatic groups such as phenyl,naphthyl, and the like, and includes substituted aryl groups such astolyl. “Halo” as used herein refers to any halogen group, such aschloro, fluoro, bromo, or iodo. The term “hydroxyalkyl” as used hereinrefers to C1 to C4 linear or branched hydroxy-substituted alkyl, i.e.,—CH₂OH, —(CH₂)₂OH, etc. The term “aminoalkyl” as used herein refers toC1 to C4 linear or branched amino-substituted alkyl, wherein the term“amino” refers to the group NR′R″, wherein R′ and R″ are independentlyselected from H or lower alkyl as defined above, i.e., —NH₂, —NHCH₃,—N(CH₃)₂, etc. The term “oxyalkyl” as used herein refers to C1 to C4oxygen-substituted alkyl, i.e., —OCH₃, and the term “oxyaryl” as usedherein refers to C3 to C10 oxygen-substituted cyclic aromatic groups.

[0017] The present invention is concerned primarily with the treatmentof human subjects, but may also be employed for the treatment of otheranimal subjects (i.e., mammals, avians) for veterinary purposes. Mammalsare preferred, with humans being particularly preferred.

[0018] The present invention is usefil in treating mucosal surfaces in asubject in need of such treatment. “Treatment” includes the hydration ofthe mucosal surface, or the blocking or retardation of the absorption ofliquid onto or into the mucosal surface, or an increase of volume ofliquid on the mucosal surface, whether by increasing water or liquid onthe mucosal surface, increasing the amount of salt on the surface, orboth. In a preferred embodiment, the mucosal surface is an airwaysurface. The term “airway surface” as used herein refers to airwaysurfaces below the larynx and in the lings (e.g., bronchial passages,alveolar passages), as well as air passages in the head, including thesinuses and other nasal airways, and in the region above the larynx. Thepresent invention may also be used to treat mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-ureteral surfaces, ocular surfaces orsurfaces of the eye, the inner ear, and the middle ear.

[0019] Subjects that may be treated by the methods of the presentinvention include patients afflicted with cystic fibrosis, primaryciliary dyskinesia, chronic bronchitis, chronic obstructive airwaydisease, artificially ventilated patients, patients with acutepneumonia, etc. Subjects that may be treated by the method of thepresent invention also include patients being nasally administeredsupplemental oxygen (which tends to dry the airway surfaces), patientsafflicted with an allergic disease or response (e.g., an allergicresponse to pollen, dust, animal hair or particles, insects or insectparticles, etc.) that affect nasal airway surfaces, patients afflictedwith an infection caused by a microorganism (e.g., infections caused bysuch organisms as Staphylococcus aureus, Haemophilits influenza,Streptococcus pneumoniae, Pseudomonas spp. etc.) of the nasal airwaysurfaces, an inflammnatory disease that affects nasal airway surfaces,or patients afflicted with sinusitis (wherein the active agent or agentsare administered to promote drainage of congested mucous secretions inthe sinuses by administering an amount effective to promote drainage ofcongested fluid in the sinuses).

[0020] The compounds of the present invention can be prepared accordingto methods described herein, as well as in accordance with knowntechniques or variations thereof which will be apparent to skilledpersons in light of the disclosure set forth herein. See, e.g., D. Benoset al., Proc. Natl. Acad. Sci. USA 83, 8525 (1986); T. Kleyman et al.,Am. J Physiol. 250 (Cell Physiol. 19): C165-C170 (1986); U.S. Pat. No.3,313,813; U.S. Pat. No. 4,501,729; U.S. Pat. No. 5,789,391; U.S. Pat.No. 5,292,498; U.S. Pat. No. 5,635,160; U.S. Pat. No. 5,837,861; U.S.Pat. No. 5,763,447; and U.S. Pat. No. 5,789,391 (the disclosures of allpatent references cited herein are incorporated by reference in theirentirety).

1. Sodium Channel Blockers

[0021] Any sodium channel blocker (i.e., P₁ or P₂ in the formulaP₁-L-P₂) can be used to carry out the present invention. Numerouspyrazinoylguanidine sodium channel blockers are disclosed in U.S. Pat.No. 3,313,813 to Cragoe. Amiloride, one particular pyrazinoylguanidinesodium channel blocker, is described at Merck Index Registry No. 426(12^(th) Ed. 1996). Benzamil (also known as3,5-diainino-6-chloro-N-(benzylaminoaminomethylene) pyrazinecarboxamide)and phenamil (also known as3,5-diamino-6-chloro-N-(phenylaminoaminomethylene)pyrazinecarboxamide)are known compounds and are also disclosed in U.S. Pat. No. 3,313,813 toE. Cragoe.

[0022] Various additional pyrazinoylguanidine sodium channel blockersthat are amiloride analogs are disclosed and described in T. Kleyman andE. Cragoe, J. Membrane Biol. 105, 1-21 (1988).

[0023] Preferred examples of active compounds that may be used to carryout the present invention are the pyrazinoylguanidine sodium channelblockers disclosed in U.S. Pat. No. 3,313,813, incorporated by referenceabove. Such compounds have the formula:

[0024] wherein:

[0025] X is selected from the group consisting of chloro, bromo, iodo,loweralkyl, lower-cycloalkyl having from 3 to 7 carbons, phenyl,chlorophenyl, bromophenyl, Z-thio and Z-sulfonyl wherein Z is selectedfrom the group consisting of loweralkyl and phenyl-loweralkyl.Preferably, X is chloro.

[0026] Y is selected from the group consisting of hydroxyl, mercapto,loweralkyloxy, loweralkylthio, chloro, loweralkyl, lowercycloalkylhaving from 3 to 6 carbons, phenyl, amino having the structure:

[0027] wherein:

[0028] R is selected from the group consisting of hydrogen, amino,amidino, lower-cycloalkyl having 3 to 6 carbon atoms, loweralkyl,hydroxyloweralkyl, halo-loweralkyl, lower-(cycloalkylalkyl) having 3 to6 carbons in the ring, phenyl-loweralkyl, lower-(alkylaminoalkyl),lower-alkenyl, phenyl, halophenyl, and lower-alkylphenyl;

[0029] R₁ is selected from the group consisting of hydrogen, loweralkyl,loweralkenyl, and additionally;

[0030] R and R₁ can be joined to form a lower alkylene. Preferably, Y isamino.

[0031] R₂ is selected from the group consisting of hydrogen andloweralkyl. Preferably, R, R₁, and R₂ are hydrogen.

[0032] R₃ and R₄ are indepenedently selected from the group consistingof hydrogen, loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl. Preferably, R₃ is hydrogen,phenyl, or phenylalkyl. Preferably, R₄ is hydrogen.

[0033] As discussed below, R₄ may be replaced with a linking group L.

2. Linking Groups

[0034] Any suitable linking group (i.e., “L” in formula P₁-L-P₂) may beemployed. The linking group may be a non-absorbable carrier moiety. Thenon-absorbable carrier moiety may be a carbohydrate, protein, peptide,polyamine, or water soluble linear polymer. Water soluble linearpolymers useful as carrier moieties include polyvinylpyrrolidone,polyethylene glycol, nonylphenol ethoxylates, and polyvinyl alcohol.Carbohydrates useful as carrier moieties include sugars andpolysaccharides, such as dextran, lactose, and mannitol. An additionalexample is agarose. Proteins or peptides useful as carrier moietiesinclude albumin (for example, human serum albumin) and protamine.Polyarnines useful for carrying out the present invention includespermine and spermidine.

[0035] The linking groups may be the same as those groups set forth forR4 above, except that they are provided in divalent rather thanunivalent form. Linking groups may also be heteroatoms, such as —O. Thusthe linking group may be an alkylene, alkylenecarbonyl,carbonylalkylene, or a carbonyl group, as follows:

[0036] where n is 0 (i.e., a direct covalent linkage) or is from 1 to 6.Such alkylene groups may be saturated or unsaturated, and may besubstituted 1, 2, 3, or 4 times with C1-C4 alkyl, halo, phenyl, orhalo-substituted phenyl. Examples are as follows:

[0037] A phenyl or phenylene group , or two or more linked phenylenegroups, may be provided as the linking group, which phenylene group mayoptionally be substituted 1, 2, or four times with a halogen or alkylgroup. Examples are as follows:

[0038] A substituted or unsubstituted phenylene group may be joined ateither or both ends with a substituted or unsubstituted alkylene,alkylenecarbonyl, carbonylalkylene, or carbonyl group as described aboveto provide a linking group. Examples are as follows:

[0039] A substituted or unsubstituted alkylene, alkylenecarbonyl,carbonylalkylene, or carbonyl group as described above may joined ateither or both ends to a substituted or unsubstituted phenylene group asdescribed above to provide a linking group. Examples are as follows:

[0040] where “n” is as defined above. Such compounds may be frthersubstituted at either or both ends by a substituted or unsubstitutedalkylene, alkylenecarbonyl, carbonylalkylene, or carbonyl group, asdescribed above, to provide still furher linking groups. Examples are asfollows:

[0041] whrere “n” is as defined above.

3. P2Y₂ Receptor Agonists

[0042] As noted above P₂ may also be a P2Y₂ receptor ligand, such as anucleotide (e.g., ATP, UTP), dinucleotide (described in more detailhereinbelow), or derivative thereof. P2Y₂ receptor ligands that can beused to carry out the present invention include all of the compounds,particularly the nucleotides and dinucleotides that are P2Y₂ ligands andare disclosed in W. Pendergast et al., U.S. Pat. No. 5,837,861 (Nov. 17,1998), along with all the compounds disclosed in U.S. Pat. Nos.5,763.447 to Jacobus and Leighton, 5,789,391 to Jacobus et al.,5,635,160 to Stutts et al., and 5,292,498 to Boucher, the disclosures ofall of which are incorporated herein by reference in their entirety.

[0043] Examples of such nucleotides are depicted in Formulae I-IV

[0044] wherein:

[0045] X₁, X₂ and X₃ are each independently either O⁻or S⁻; preferably,X₂ and X₃ are O;

[0046] R₁ is O, imido, methylene or dihalomethylene (e.g.,dichloromethylene or difluoromethylene); preferably, R₁ is oxygen ordifluoromethylene;

[0047] R₂ is H or Br; preferably, R₂ is-H; particularly preferredcompounds of Formula I are uridine 5′-triphosphate (UTP) and uridine5′-O-(3-thiotriphosphate) (UTPγS).

[0048] A dinucleotide is depicted by the general Formula II:

[0049] wherein:

[0050] X is oxygen, methylene, difluoromethylene, imido;

[0051] n=0, 1, or 2;

[0052] m=0, 1, or 2;

[0053] n+m=0, 1, 2, 3,or 4;and

[0054] B and B′ are each independently a purine residue or a pyrimidineresidue linked through the 9- or 1-position, respectively;

[0055] Z=OH or N₃;

[0056] Z′=OH or N₃;

[0057] Y=H or OH;

[0058] Y′=H or OH;

[0059] provided that when Z is N₃, Y is H or when Z′ is N₃, Y′ is H.

[0060] The furanose sugar is preferably in the β-configuration.

[0061] The furanose sugar is most preferably in the β-D-configuration.

[0062] Preferred compounds of Formula II are the compounds of FormulaIIa:

[0063] wherein:

[0064] X=O;

[0065] n+m=1 or 2;

[0066] Z, Z′, Y, and Y′=OH;

[0067] B and B′ are defined in Formulas IIc and IId;

[0068] X=O

[0069] n+m=3 or 4;

[0070] Z, Z′, Y, and Y′=OH;

[0071] B=uracil;

[0072] B′ is defined in Formulas IIc and IId; or

[0073] X=O;

[0074] n+m=l or 2;

[0075] Z, Y, and Y′=OH;

[0076] Z′=H;

[0077] B=uracil;

[0078] B′ is defined in Formulas IIc and IId; or

[0079] X=O;

[0080] n+m−0, 1, or 2;

[0081] Z and Y=OH;

[0082] Z′=N₃;

[0083] Y′=H;

[0084] B=uracil;

[0085] B′=tymine; or

[0086] X=O;

[0087] n+m-=0, 1, or 2;

[0088] Z and Z′=N₃;

[0089] Y and Y′=H;

[0090] B and B′=thymine; or

[0091] X=CH₂, CF₂, or NH;

[0092] n and m=1;

[0093] Z, Z′, Y, and Y′=OH;

[0094] B and B′ are defined in Formulas Ic and IId.

[0095] Another preferred group of the compounds of Formula II are thecompounds of Formula IIb or the pharmaceutically acceptable saltsthereof:

[0096] wherein:

[0097] X is oxygen, methylene, difluoromethylene, or imido;

[0098] n=0 or 1;

[0099] m=0 or 1;

[0100] n+m=0, 1, or 2; and

[0101] B and B′ are each independently a purine residue, as in FormulaIIc, or a pyrimidine residue, as in Formula IId, linked through the 9-or 1-position, respectively. In the instance where B and B′ are uracil,attached at N-1 position to the ribosyl moiety, then the total of m+nmay equal 3 or 4 when X is oxygen. The ribosyl moieties are in theD-configuration, as shown, but may be L-, or D- and L-. TheD-configuration is preferred.

[0102] The substituted derivatives of adenine include adenine 1-oxide;1,N⁶-(4- or 5-substituted etheno) adenine; 6-substituted adenine; or8-substituted aminoadenine, where R′ of the 6- or 8-HNR′ groups arechosen from among: arylalkyl (C₁₋₆) groups with the aryl moietyoptionally functionalized as described below; alkyl; and alkyl groupswith functional groups therein, such as:([6-aminohexyl]carbamoylmethyl)-, and ω-acylated-amino(hydroxy, thioland carboxy) derivatives where the acyl group is chosen from among, butnot limited to, acetyl, trifluroroacetyl, benzoyl, substituted-benzoyl,etc., or the carboxylic moiety is present as its ester or amidederivative, for example, the ethyl or methyl ester or its methyl, ethylor benzamido derivative. The ω-amino(hydroxy, thiol) moiety may bealkylated with a C₁₋₄ alkyl group.

[0103] Likewise, B or B′ or both in Formula IIb may be a pyrimidine withthe general formula of FIG. IId, linked through the 1-position:

[0104] wherein:

[0105] R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkoxy, C₁₋₆ alkylamino, and dialkylamino, the alkyl groups optionally linked toform a heterocycle;

[0106] R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅ alkanoyl, benzoyl,or sulphonate;

[0107] R₆ is hydroxy, mercapto, alkoxy, aralkoxy, C₁₋₆-alkylthio, C₁₋₅,disubstituted amino, triazolyl, alkylamino, or dialkylamino, where thealkyl groups are optionally linked to form a heterocycle or linked toN-3 to form an optionally substituted ring;

[0108] R₇ is hydrogen, hydroxy, cyano, nitro, alkenyl, with the alkenylmoiety optionally linked through oxygen to form a ring optionallysubstituted on the carbon adjacent to the oxygen with alkyl or arylgroups, substituted alkynyl or hydrogen where R₈ is amino or substitutedamino and halogen, alkyl, substituted alkyl, perhalomethyl (e.g., CF₃),C₂₋₆ alkyl, C₂₋₃ alkenyl, or substituted ethenyl (e.g., allylamino,bromvinyl and ethyl propenoate, or propenoic acid), C₂₋₃ alkynyl orsubstituted alkynyl when R6 is other than amino or substituted amino andtogether R₅-R₆ may form a 5- or 6-membered saturated or unsaturated ringbonded through N or O at R₆, such a ring may contain substituents thatthemselves contain functionalities;

[0109] R₈ is hydrogen, alkoxy, arylalkoxy, alkylthio, arylalkylthio,carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy, orphenylthio.

[0110] In the general structure of FIG. IId above, the dotted lines inthe 2- to 6-positions are intended to indicate the presence of single ordouble bonds in these positions; the relative positions of the double orsingle bonds being determined by whether the R₄, R₆, and R₇ substituentsare capable of keto-enol tautomerism.

[0111] In the general structures of FIG. IIc and IId above, the acylgroups advantageously comprise alkanoyl or aroyl groups. The alkylgroups advantageously contain 1 to 8 carbon atoms, particularly 1 to 4carbon atoms optionally substituted by one or more appropriatesubstituents, as described below. The aryl groups including the arylmoieties of such groups as aryloxy are preferably phenyl groupsoptionally substituted by one or more appropriate substituents, asdescribed below. The above mentioned alkenyl and alkynyl groupsadvantageously contain 2 to 8 carbon atoms, particularly 2 to 6 carbonatoms, e.g., ethenyl or ethynyl, optionally substituted by one or moreappropriate substituents as described below. Appropriate siibstituentson the above-mentioned alkyl, alkenyl, alkynyl, and aryl groups areadvantageously selected from halogen, hydroxy, C₁₋₄ alkoxy, C₁₋₄ alkyl,C₆₋₁₂ arylalkoxy, carboxy, cyano, nitro, sulfonamido, sulfonate,phosphate, sulfonic, amino, and substituted amino wherein the amino issingly or doubly substituted by a C₁₋₄ alkyl, and when doublysubstituted, the alkyl groups optionally being linked to form aheterocycle.

[0112] For purposes of further clarifying the foregoing descriptions ofFormulae IIc and IId, the descriptions can be simplified to thefollowing:

[0113] R₂ is O or is absent; or

[0114] R₁ and R₂ taken together may form optionally substituted5-membered fused imidazole ring; or

[0115] R₁ of the 6-HNR₁ group or R₃ of the 8-HNR₃ group is chosen fromthe group consisting of:

[0116] (a) arylalkyl (C₁₋₆) groups with the aryl moiety optionallysubstituted,

[0117] (b) alkyl,

[0118] (c) ([6-aminohexyl]carbamoylmethyl),

[0119] (d) ω-arnino alkyl (C₂₋₁₀),

[0120] (e) ω-hydroxy alkyl (C₂₋₁₀),

[0121] (f) ω-thiol alkyl (C₂₋₁₀),

[0122] (g) ω-carboxy alkyl (C₂₋₁₀),

[0123] (h) the ω-acylated derivatives of (b), (c) or (d) wherein theacyl group is either acetyl, trifluroacetyl, benzoyl, or substituted-benzoyl alkyl(C₂₋₁₀), and

[0124] (i) ω-carboxy alkyl (C₂₋₁₀) as in (e) above wherein thecarboxylic moiety is an ester or an amide;

[0125] wherein:

[0126] R₄ is hydroxy, mercapto, amino, cyano, aralkoxy, C₁₋₆ alkylthio,C₁₋₆ alkoxy, C₁₋₆ alkylamino or dialkylamino, wherein the alkyl groupsof said dialkylamino are optionally linked to form a heterocycle;

[0127] R₅ is hydrogen, acyl, C₁₋₆ alkyl, aroyl, C₁₋₅ alkanoyl, benzoyl,or sulphonate;

[0128] R₆ is hydroxy, mercapto, alkoxy, aralkoxy, C₁₋₆-alkylthio, C₁₋₅disubstituted amino, triazolyl, alkylamino or dialkylamino, wherein thealkyl groups of said dialkylamino are optionally linked to form aheterocycle or linked to N³ to form an optionally substituted ring;

[0129] R₅-R₆ together forms a 5 or 6-membered saturated or unsaturatedring bonded through N or O at R₆, wherein said ring is optionallysubstituted;

[0130] R₇ is selected from the group consisting of:

[0131] (a) hydrogen,

[0132] (b) hydroxy,

[0133] (c) cyano,

[0134] (d) nitro,

[0135] (e) alkenyl, wherein the alkenyl moiety is optionally linkedthrough oxygen to form a ring optionally substituted with alkyl or arylgroups on the carbon adjacent to the oxygen,

[0136] (f) substituted alkynyl

[0137] (g) halogen,

[0138] (h) alkyl,

[0139] (i) substituted alkyl,

[0140] (j) perhalomethyl,

[0141] (k) C₂₋₆ alkyl,

[0142] (l) C₂₋₃ alkenyl,

[0143] (m) substituted ethenyl,

[0144] (n) C₂₋₃ alkynyl and

[0145] (o) substituted alkynyl when R₆ is other than amino orsubstituted amino;

[0146] R₈ is selected from the group consisting of:

[0147] (a) hydrogen,

[0148] (b) alkoxy,

[0149] (c) arylalkoxy,

[0150] (d) alkylthio,

[0151] (e) arylalkylthio,

[0152] (f) carboxamidomethyl,

[0153] (g) carboxymethyl,

[0154] (h) methoxy,

[0155] (i) methylthio,

[0156] Q) phenoxy and

[0157] (k) phenylthio.

[0158] CTP and its analogs are depicted by general Formula III:

[0159] wherein:

[0160] R₁, X₁, X₂ and X₃ are defined as in Formula I;

[0161] R₅ and R₆ are H while R₇ is nothing and there is a double bondbetween N-3 and C-4 (cytosine), or

[0162] R₅, R₆ and R₇ taken together are —CH═CH—, forming a ring from N-3to N-4 with a double bond between N-4 and C-4 (3,N⁴-ethenocytosine)optionally substituted at the 4- or 5-position of the etheno ring.

[0163] ATP and its analogs are depicted by general Formula IV:

[0164] wherein:

[0165] R₁, X₁, X₂, and X₃ are defined as in Formula I;

[0166] R₃ and R₄ are H while R₂ is nothing and there is a double bondbetween N-1 and C-6 (adenine), or

[0167] R₃ and R₄ are H while R₂ is O and there is a double bond betweenN-1 and C-6 (adenine 1 -oxide), or

[0168] R₃, R₄, and R₂ taken together are —CH═CH—, forming a ring fromN-6 to N-1 with a double bond between N-6 and C-6 (1N6-ethenoadenine).

[0169] For simplicity, Formulas I, II, III, and IV herein illustrate theactive compounds in the naturally occurring D-configuration, but thepresent invention also encompasses compounds in the L-configuration, andmixtures of compounds in the D-and L-configurations, unless otherwisespecified. The naturally occurring D-configuration is preferred.

[0170] Some compounds of Formulas I, II, III, and IV can be made bymethods which are well known to those skilled in the art and inaccordance with known procedures (Zamecnik, P., et al., Proc. Natl Acad.Sci. USA 89:2370-2373 (1992); Ng, K., et al., Nucleic Acids Res.15:3572-3580 (1977); Jacobus, K. M., et al., U.S. Pat. No. 5,789,391 andPendergast, W., et al., International Patent Application WO98/34942));some are commercially available, for example, from Sigma ChemicalCompany, PO Box 14508, St. Louis, Mo. 63178. The synthetic methods ofU.S. Pat. No. 5,789,391 and International Patent Application WO98/34942are incorporated herein by reference in their entirety.

[0171] Thus, examples of compounds that can be used to carry out thepresent invention include compounds of Formula I-IV above, and includecompounds having the general formula:

[0172] wherein:

[0173] X may be O or S;

[0174] A is a purine or pyrimidine base (e.g., adenine, guanine,tlhymine, cytosine, uracil)(each purine or pyrimidine base is preferablyjoined to the ribose or deoxyribose ring by covalent bond to the 9nitrogen in the case of purines, or by covalent bond to the 1 nitrogenin the case of pyrimidines);

[0175] R₁ is H or OH; and

[0176] n is from 1 to 4or 6,preferably 2, 3 or 4.

[0177] Additional examples of receptor agonists that can be used tocarry out the present invention are dinucleotides, including thosehaving the general formula:

[0178] wherein:

[0179] A and B are each independently a purine or pyrimidine base (e.g.,adenine, guanine, thymine, cytosine, uracil); preferably, A is uraciland B is cytosine;

[0180] R₁ and R₂ are each independently selected from the groupconsisting of H or OH; and n is from 1 to 6, preferably 3 or 4.

[0181] For P2Y₂ receptor ligands as described herein, the linking groupmay be covalently joined to the purine or pyrimidine base, or thecorresponding ribose or deoxyribose ring (e.g., of the compounds ofFormula I-IV above), or attached to the terminal phosphate moiety ofcompounds represented by Formulae I, II and IV above, by any suitablemeans, such as by covalently joining the linking group thereto in anysuitable position (e.g., a ring carbon such as the 5 carbon in apyrimidine, or the 2, 6 or 8 carbon in a purine), to which linking groupthe ligand may be covalently attached.

4. Example Conjugate Compounds

[0182] Specific examples of active compounds of the present invention,where P₂ is a pyrazinoylguanidine sodium channel blocker, include butare not limited to the following:

[0183] Additional examples of conjugate compounds useful in the presentinvention include those compounds whose structures are shown in Table 1,below, and in the Examples that follow.

[0184] Examples of active compounds of the present invention, where P₂is a P2Y₂ receptor ligand, are as follows:

5. Pharmaceutically Acceptable Salts

[0185] The term “active agent” as used herein, includes thepharmaceutically acceptable salts of the compound, such as (but notlimited to) benzamil hydrochloride or phenamil hydrochloride.Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects. Examples of such salts are (a) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuiric acid, phosphoric acid, nitric acid and thelike; and salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid. p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0186] For nucleotides or dinucleotide active compounds, the compoundsmay be prepared as an alkali metal salt such as sodium or potassium, analkaline earth metal salt, or an ammonium and tetraalkyl ammonium salt,NX4⁺ (wherein X is a C₁₋₄) alkyl group. Pharmaceutically acceptablesalts are salts that retain the desired biological activity of theparent compound and do not impart undesired toxicological effects.

[0187] Active agents used to prepare compositions for the presentinvention may alternatively be in the form of a pharmaceuticallyacceptable free base of active agent. Because the free base of thecompound is less soluble than the salt, free base compositions areemployed to provide more sustained release of active agent to the lungs.Active agent present in the lungs in particulate form which has not goneinto solution is not available to induce a physiological response, butserves as a depot of bioavailable drug which gradually goes intosolution.

6. Formulations and Administration

[0188] A third aspect of the present invention is a pharmaceuticalformulation, comprising an active compound as described above in apharmaceutically acceptable carrier (e.g., an aqueous carrier solution).In general, the active compound is included in the composition in anamount effective to treat mucosal surfaces, such as inhibit thereabsorption of water by airway surfaces, including nasal airwaysurfaces.

[0189] The active compounds disclosed herein may be administered tomucosal surfaces by any suitable means, including topically,parenterally (e.g., by intraveneous, intramuscular, or intraperitonealinjection), orally, rectally, via inhalation, transdermally, etc. Forexample, for the treatment of constipation, the active compounds may beadministered orally or rectally to the gastrointestinal mucosal surface.The active compound may be combined with a pharmaceutically acceptablecarrier in any suitable form, such as sterile physiological saline foran injectable or topical solution, as a droplet, tablet or the like fororal administration, as a suppository for rectal or genito-ureteraladministration, etc. Excipients may be included in the formulation toenhance the solubility of the active compounds, as desired.

[0190] The active compounds disclosed herein may be administered to theairway surfaces of a patient by any suitable means, including as aspray, mist, or droplets of the active compounds in a pharmaceuticallyacceptable carrier such as physiological saline solution or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

[0191] In one preferred embodiment they are administered byadministering an aerosol suspension of respirable or non-respirableparticles (preferably non-respirable particles) comprised of the activecompound, which the subject inhales through the nose. The respirable ornon-respirable particles may be liquid or solid. The quantity of activeagent included may be an amount sufficient to achieve dissolvedconcentrations of active agent on the airway surfaces of the subject offrom about 10⁻⁹, 10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², or 10⁻¹ Moles/liter,and more preferably from about 10⁻⁶ to about 10⁻⁴ Moles/liter.

[0192] In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt such as benzamil hydrochloride orphenamil hydrochloride to provide both early release of and sustainedrelease of active agent for dissolution into the mucous secretions ofthe nose. Such a composition serves to provide both early relief to thepatient, and sustained relief over time. Sustained relief, by decreasingthe number of daily administrations required, is expected to increasepatient compliance with a course of active agent treatments.

[0193] Solid or liquid particulate active agent prepared for practicingthe present invention should as noted above include particles ofrespirable or non-respirable size: that is, for respirable particles,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs,and for nonrespirable particles, particles sufficiently large to beretained in the nasal airway passages rather than pass through thelarynx and into the bronchi and alveoli of the lungs. In general,particles ranging from about 1 to 5 microns in size (more particularly,less than about 4.7 microns in size) are respirable. Particles ofnon-respirable size are greater than about 5 microns in size, up to thesize of visible droplets. Thus, for nasal administration, a particlesize in the range of 10-500 μm may be used to ensure retention in thenasal cavity.

[0194] The dosage of active compound will vary depending on thecondition being treated and the state of the subject, but generally maybe an amount sufficient to achieve dissolved concentrations of activecompound on the nasal airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², or 10⁻¹ Moles/liter, and morepreferably from about 10⁻⁶ to about 3×10⁻⁴ Moles/liter. Depending uponthe solubility of the particular formulation of active compoundadministered, the daily dose may be divided among one or several unitdose administrations. The daily dose by weight may range from about 0.1,0.5 or 1 to 10 or 20 milligrams of active agent particles for a humansubject, depending upon the age and condition of the subject. Acurrently preferred unit dose is about 0.005 milligrams of active agentgiven at a regimen of four administrations per day. The dosage may beprovided as a prepackaged unit by any suitable means (e.g.,encapsulating in a gelatin capsule).

[0195] Pharmaceutical formulations suitable for airway administrationinclude formulations of solutions, emulsions, suspensions and extracts.See generally, J. Naim, Solutions, Emulsions, Suspensions and Extracts,in Remington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed1995). Pharmaceutical formulations suitable for nasal administration maybe prepared as described in U.S. Pat. Nos. 4,389,393 to Schor; 5,707,644to Ilium; 4,294,829 to Suzuki; and 4,835,142 to Suzuki; the disclosuresof which are incorporated by reference herein in their entirety.

[0196] In the manufacture of a formulation according to the invention,active agents or the physiologically acceptable salts or free basesthereof are typically admixed with, inter alia, an acceptable carrier.The carrier must, of course, be acceptable in the sense of beingcompatible with any other ingredients in the formulation and must not bedeleterious to the patient. The carrier may be a solid or a liquid, orboth, and is preferably formulated with the compound as a unit-doseformulation, for example, a capsule, which may contain from 0.5% to 99%by weight of the active compound. One or more active compounds may beincorporated in the formulations of the invention, which formulationsmay be prepared by any of the well-known techniques of pharmacyconsisting essentially of admixing the components.

[0197] Mists or aerosols of liquid particles comprising the activecompound may be produced by any suitable means, such as by a simplenasal spray with the active agent in an aqueous pharmaceuticallyacceptable carrier, such as sterile saline solution or sterile water.Administration may be with a pressure-driven aerosol nebulizer or anultrasonic nebulizer. See, e.g., U.S. Pat. Nos. 4,501,729 and 5,656,256.Suitable formulations for use in a nasal droplet or spray bottle or innebulizers consist of the active ingredient in a liquid carrier, theactive ingredient comprising up to 40% w/w of the formulation, butpreferably less than 20% w/w. The carrier is typically water (and mostpreferably sterile, pyrogen-free water) or a dilute aqueous alcoholicsolution, preferably made isotonic with body fluids by the addition of,for example, sodium chloride. Optional additives include preservativesif the formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents and surfactants.

[0198] Mists or aerosols of solid particles comprising the activecompound may likewise be produced with any solid particulate medicamentaerosol generator. Aerosol generators for administering solidparticulate medicaments to a subject produce particles which arerespirable or non-respirable, as explained above, and generate a volumeof mist or aerosol containing a predetermined metered dose of amedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained. in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of a powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises from 0.1 to 100 w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofthe active ingredient in a liquified propellant. During use thesedevices discharge the formulation through a valve adapted to deliver ametered volume, typically from 10 to 150 or 200 μl to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation mayadditionally contain one or more co-solvents, for example, ethanol,surfactants (such as oleic acid or sorbitan trioleate), antioxidants andsuitable flavoring agents.

[0199] Compositions containing respirable or non-respirable dryparticles of micronized active agent may be prepared by grinding the dryactive agent with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates.

[0200] The particulate active agent composition may optionally contain adispersant which serves to facilitate the formation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

7. Covalent Conjugate of AA Pyrazinoylguanidine Sodium Channel Blockerand a Non-absorbable Carrier Moiety

[0201] In addition to the foregoing, the pyrazinoylguanidine sodiumchannel blockers described above can be conjugated to a non-absorbablecarrier moiety as described above to provide compounds that are activein hydrating mucosal surfaces. Some of these compounds have the formula:

[0202] wherein: X, Y, R₂ and R₃ are as defined above, and Z is anon-absorbable carrier moiety as described above covalently linked tothe adjacent nitrogen atom; or a pharmaceutically acceptable saltthereof. Such compounds can be prepared, formulated and administered inessentially the same manner as described above for the same uses asdescribed above.

[0203] The following Examples are provided to illustrate the presentinvention, and should not be construed as limiting thereof. In theseExamples, Proton NMR spectra (360 MHz) and carbon NMR spectra (90 MHz)were obtained on a Bruker WM-360 spectrometer using tetramethylsilane asan internal standard. Liquid chromatography (LC)/mass spectroscopy (MS)was performed on a Perkin Elmer Sciex API 100 by one of the followingmethods:

[0204] Method A: YMC Pro C8 column, 5μ, 150×4.6 mm; Mobile phase A=water+0.4% acetic acid, B=acetonitrile (MeCN)+0.4% acetic acid; Gradient: 5%B for 1 min, to 80% B in 7 min. followed by 100% B for 5 min.

[0205] Method B: YMC Pro C8 column, 5μ, 150×4.6 mm; Mobile Phase A=water+0.4% acetic acid, B=MeCN+0.4% acetic acid; Gradient: 5% B for 1 min,going up to 80% B in 5 min.

[0206] Method C: Luna C8 (2) column, 150×4.6 mm, 5μ, detector λ=360 nm,mobile phase A=water+0.4% acetic acid, B=MeCN+0.4% acetic acid;Gradient: 5% B for 1 min, to 8 0% B in 7 min, followed by washout with100% B for 5 min.

[0207] Analytical HPLC was performed on a Shimadzu HPLC 10Avp by one ofthe following methods:

[0208] Method D: Luna C18(2) column, 5μ, 250×4.6 mm; detector λ=360 nm;Gradient: A=water+0.1% trifluoroacetic acid(TFA), B=MeCN+0.1% TFA,concentration of MeCN increases from 10 to 60% during a 0-11 mininterval, then 60-100% from 11-12 min.

[0209] Method E: Symmetry C8 column, 150×4.6 mm; detector λ=360 nm;Gradient: A=water+0.1% TFA, b=MeCN+0.1% TFA, concentration of Bincreases in the A/B mixture from 10 to 60% during the 0-11 mininterval, then B increases to 60-100% from 11-12 min.

[0210] Preparative HPLC was performed on a Gilson CombiChem by methodsdescribed in below in the Examples.

EXAMPLE 1 Synthesis of Dimeric Compounds

[0211] Referring to Scheme 1, dimer compounds of Formula I have beensynthesized as shown in TABLE 1. The synthesis begins with1-(3,5-diamino-6-chloropyrazinoyl)-2-methyl-2-thiopseudourea hydroiodide(intermediate II, prepared as described in U.S. Pat. No. 4,246,406 toCragoe et al).. Intermediate II was treated withN-(benzyloxycarbonyloxy)succinimide and triethylamnine inN,N-dimethylformamide (DMF) to give the carbobenzyloxy(Cbz)-protectedintermediate III. Formation of the Cbz-protected dimer V occurs upontreatment of intermediate III with the appropriate diamine IV -in thepresence of mercury(II) chloride and triethylamine in DMF (conditionsreported by W. Su, Synth. Comm., 26, 407-413 (1996) for the preparationof Cbz-protected guanidines). Treatment of dimer intermediate V withhydrobromic acid in acetic acid removes both of the Cbz-protectinggroups to give amiloride dimer I as the dihydrobromide salt. Thehydrobromide salts of I could be converted to the free base of I bytreatment with a strong base like potassium hydroxide in aqueous media.The free base can then be converted to other salt forms (e.g,.hydrochloride salt or other pharmaceutically acceptable salt forms) bytreatment with the appropriate acid. TABLE 1 Dimers Max Effect ofInhibition washout (3) Compound IC50 (%) from 100% = (AMR code)Structure (nM) baseline Baseline CF-509 1363

1275 79 77 CF-510 1390

81 73 21 CF-511 1438

114 86 54 CF-512 1465

197 92 60 CF-514 1504A

121 96 20 CF-515 1504

133 99 12 CF-516 1527

330 90 65 *CF-519 1604

1075 85 100

[0212]

EXAMPLES 2 THROUGH 10 Preparation of Dimeric Analogues of AmilorideEXAMPLE 2

[0213] N-Cbz-1-(3,5-diamino-6-chloropyrazinol)-2-methyl-pseudothiourea(III)

[0214] 1-(3,5-Diamino-6-chloropyrazinoyl)-2-methyl-pseudothioureahydroiodide (II, 494 mg, 1.27 mmol) was dissolved in a mixture ofanhydrous DMF (10 mL), and triethylamine (3 mL) followed by treatmentwith N-(benzyloxycarbonyloxy) succinimide (470 mg, 1.7 mmol) dissolvedin DMF (3 mL). The reaction mixture was stirred overnight at roomtemperature. After this time, the reaction mixture was concentratedunder reduced pressure and the residue suspended in ethyl acetate (30mL). Silica gel (25 g) was added to the solution and the solvent wasevaporated to leave the silica gel impregnated with the crude productthat was purified by flash chromatography on a FlashElute™ system fromElution Solution (P.O. Box 5147, Charlottesville, Va. 22905) using a 90g silica gel cartridge (eluent: hexanes, ethyl acetate=1:2). ThepurifiedN-Cbz-1-(3,5-diamino-6-chloropyrazinoyl)-2-methyl-pseudothiourea (III)was obtained as a pale yellow solid: 416 mg (83% yield); ¹H NMR (360MHz, DMSO-d₆) δ2.33 (s, 3H), 2.61 (s, 3H), 4.99 (s, 2H), 7.39 (m, 10H),13.7 (s, 1H); API MS m/z=395 [C₁₅H₁₅ClN₆O₃S+H]⁺; LC/MS (Method A)>99%,t_(r)=10.1 min.

EXAMPLE 3

[0215] 1,5-Bis[3,5-diamino-6-chloropyrazinoyl)guanidinol-3-oxa-pentaneDihydrobromide (Ia)

[0216] A solution of 1,5-diamino-3-oxa-pentane (IVa, 30 μL, 0.3 =mol) indry DMF (100 μL) was added to intermediate III (226 mg, 0.6 mmol) andstirred in anhydrous DMF (10 mL). Triethylamine (480 μL, 3.4 mmol) inDMF (1 mL) and mercury(II) chloride (154 mg, 0.6 mmol) in DMF (100 μL)were added and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was filtered through silica gel andthe filtrate concentrated under reduced pressure. The residue waspurified by flash chromatography on FlashElute™ system from ElutionSolution using 90 g silica gel cartridge (eluent: ethyl acetate,hexanes=7:1). The fractions were analyzed by LC/MS (Method B) and thosefractions containing the desired product were combined and concentratedto give1,5-bis[(N-Cbz-3,5-diamino-6-chloropyrazinoyl)guanidino]-3-oxa-pentane(Va, 102 mg, 44% yield); LC/MS >99% Method B); API MS m/z=797[C₃₂H₃₄Cl₂N₁₄O₇+H]⁺.

[0217] Intermediate Va (50 mg) was dissolved in 30% HBr in acetic acid(10 mL) and the mixture was stirred for 2 d. The volume of the reactionmixture was reduced (to 4 mL) when a precipitate formed. Ethyl ether (10mL) was added to the acetic acid/product mixture and the precipitate wascollected by vacuum filtration, washed with additional ether, dried andthen purified by preparative HPLC on Luna column [C18(2), 5μ, 250×21.2mm; mobile phase MeCN/water containing 0.1% TFA; gradient: 5% MeCN fromthe 0-2 min interval, increased from 5% -40% MeCN from 2-10 min, 40%-80% MeCN from 10-19 min, 40% -80% MeCN from 19-23 min, 80% -100% MeCNand 100% MeCN from 23-25 min.] Fractions containing the target compoundwere combined, concentrated under reduced pressure and the residueredissolved in 10% HBr and evaporated to dryness and washed with THF.The product Ia was obtained as a yellow powder: 18.9 mg (41% yield fromV); ¹H NMR (360 MHz, DMF-d₇) δ3.80 (m, 4H), 3.88 (m, 4H), 7.51 (br s,4H), 9.58 (m, 2H), 10.97 (s, 2H). API MS m/z=529 [C₁₆H₂₂Cl₂N₁₄O₃+H]⁺;HPLC (Method D) >99%, t_(r)=6.72 min.

EXAMPLE 4

[0218] 1,4-Bisf(3,5-diamino-6-chloropyrazinoyl)guanidino]butanedihydrobromide (Ib)

[0219] A solution of 1,4-diaminobutane (IVb, 24 mg, 0.3 nmuol) in dryDMF (230 μL) was added to III (213 mg, 0.54 mmol) in anhydrous DMF (10mL), followed by addition of triethylarnine (480 μL, 3.4 mmol) in DMF (1mL) and mercury(II) chloride (146 mg, 0.53 mmol) in DMF (600 μL). Thereaction mixture was stirred for 3 d at room temperature, then filteredthrough silica gel. The filtrate was concentrated under reduced pressureand the residue was dissolved in 30% HBr in acetic acid (20 mL) andstirred overnight at rt. The reaction mixture was poured into ethylether (150 mL) resulting in the formation of a precipitate that wasisolated by vaculun filtration and washed with water (3×0.5 mL). Thesolid precipitate was purified by preparative HPLC on a Luna C18(2)column [5μ, 250×21.2 mm; flow rate=20 mL/min; mobile phase consists ofMeCN/water containing 0.1% TFA; gradient: 10% MeCN from the 0-2 mininterval, concentration of MeCN increased from 10% -40% from 2-10 min,40% -100% MeCN from 10-19 min, 100% MeCN from 19-23 min, MeCN decreasedfrom 100% -10% from 23-25 min]. Fractions containing the target compoundwere combined and concentrated under reduced pressure to give a residuethat was redissolved in 10% HBr and evaporated to dryness and washedwith ethyl ether to give Ib as a pale yellow solid: 19.4 mg (10.1%yield); ¹HNMR (360 MHz, DMSO-d₆) δ1.62 (br s, 4H), 7.43 (s, 4H), 8.77(br S, 2H), 8.89 (br s, 2H), 9.24 (s, 2H), 10.48 (s, 2M); ¹³C NMR (90MHz, DMSO-d₆) δ24.8, 40.4, 108.9, 119.5, 153.1, 154.2, 155.9 and 165.1;API MS=513 [C₁₆H₂₂Cl₂N₁₄O₂+H]⁺; HPLC (Method D) >99%, t_(r) 6.26 min.

EXAMPLE 5

[0220] 1,5-Bis[(3,5-diamino-6-chloropyrazinoyl)guanidino]hexaneDihydrobromide (Ic)

[0221] Compound Ic was prepared following the same procedure describedfor Ib. The Cbz-protected pseudothiourea III (226 mg, 0.6 mmol) and1,6-diaminohexane (IVc, 34.9 mg, 0.3 mmol) reacted in the presence oftriethylamine (480 μL, 3.4 mmol) and mercury(II) chloride (162.9 mg, 0.6mmol) to give the crude intermediate Vc, which was treated with 30% HBrin acetic acid as previously described. The crude product was purifiedby preparative HPLC on a Luna C18(2) column [5μ, 250×21.2 mm; flowrate=20 mL/min; mobile phase: MeCN/water (containing 0.1% TFA);gradient: 15% MeCN for 0-2 min interval, increase concentration of MeCNfrom 15% -30% from 2-10 min, 30% -50% MeCN from 10-19 min, 50% -100%MeCN from 19-23 min, then decrease concentration MeCN from 100% -15%from 23-25 min]. Fractions containing the target compound were combinedand concentrated under reduced pressure to give a residue that wasredissolved in 10% HBr and evaporated to dryness and washed with ethylether to give Ic: 28.8 mg (13.5% yield based on III); ¹H NMR (360 MHz,DMSO-d₆,) δ1.38 (br s, 4H), 1.59(br s, 4H), 3.38 (m, 2H), 7.44 (s, 4H),8.75 (br s, 2H), 8.90 (br s, 2H), 9.19 (s, 2H) and 10.47 (s, 2H); ¹³CNMR (90 MHz, DMSO-d₆,) δ25.5, 27.5, 40.9, 108.9, 119.6, 153.1, 154.2,155.8 and 165.1; API MS m/z=541 [C₁₈H₂₆Cl₂N₁₄O₂+H]⁺; HPLC (Method D)95.2%, t_(r)=7.26 min.

EXAMPLE 6

[0222] 1,3-Bis[(3,5-diamino-6-chloropyrazinoy)guanidino]xyleneDihydrobromide (Id)

[0223] Compound Id was prepared following the same procedure describedfor Ib. Triethylamine (480 μL, 3.4 mmol) and mercury(II) chloride (192mg, 0.7 mmol) were added to a solution of Cbz-protected pseudothioureaIII (280 mg, 0.7 rnmol) and 1,3-xylylene diamine (IVd, 50 mg, 0.3 mmol)in DMF (30 mL). The reaction mixture was stirred at rt for 48 h andworked up the same as in the procedure for Ib and followed by treatmentof the crude intermediate Vd with 30% HBr in acetic acid as previouslydescribed. The resulting crude product (yellow solid) was crystallizedfrom methanol and further purified by preparative HPLC on a symmetry C8column [7μ, 200×40 mm, flow rate=40 mL/min; mobile phase: MeCN/water(containing 0.1% TFA); gradient: concentration of MeCN 5% for 0-2 mininterval, then increased from 5% -20% MeCN from 2-10 min, 20% -60% MeCNfrom 10-30 min, 60% -100% MeCN from 30-33 min and concentrationdecreased from 100% -5% MeCN from 33-35 min]. Product isolation andfurther treatment with HBr as previously described gave the product Idas pale yellow solid: 31.2 mg (12.1% yield from III); ¹H NMR (360 MHz,DMSO-d₆,) δ4.60 (d, J=5.2 Hz, 4H), 7.40-7.42 (m, 7h), 9.03 (br s, 4H),9.61 (s, 2H) and 10.59 (s, 2H); API MS m/z=561 [C₂₀H₂₂Cl₂N₁₄O+H]⁺; HPLC(Method E) 97.3%, t_(r)=5.5 min.

EXAMPLE 7

[0224] 1,5-Bis[(35-diamino-6-chloropyrazinoyl)guanidino]pentaneDihydrobromide (Ie)

[0225] Compound Ie was prepared following the same procedure describedfor Ib. The Cbz-protected pseudothiourea III (280 mg, 0.7 mmol) and1,5-diamninopentane (IVe, 37 mg, 0.35 mmol) were reacted in the presenceof triethylamine (480 μL, 3.4 mmol) and mercury(Il) chloride (I 92 mg,0.7 mmol). The reaction mixture was stirred at rt for 24 h and worked upthe same as in the procedure for Ib and the resulting crude intermediateVe was treated with 30% HBr in acetic acid for 24 h as previouslydescribed. The reaction mixture was poured into ethyl ether (200 mL),the precipitate was collected by filtration, washed with ether, THF andthen crystallized twice from 12% HBr to give crude Ie (117 mg, 87%purity, 47% yield from III) as a yellow solid. A portion of thismaterial (78 mg) was crystallized again from 12% HBr to give Ie as apale yellow solid: 32 mg (12.8% yield from III); ¹H NMR (360 MHz,DMSO-d₆,) δ1.39 (m, 2H), 1.61 (m, 4H), 3.31 (m, 4H), 7.44 (br s, 4H),8.72 (br s, 2H), 8.90 (br s, 2H), 9.20,(s, 2H) and 10.49 (s, 2H); ¹³CNMR (90 MHz, DMSO-d₆) δ23.1,27.3, 40.8, 109.0, 119.7, 153.1, 154.2,155.9 and 165.2; API MS m/z=527 [C₁₇H₂₄Cl₂N₁₄O₂+H]⁺; HPLC (Method E)95.3%, t_(r)=5.72 min.

EXAMPLE 8

[0226] 1,5-Bis[(3,5-diamino-6-chloropyrazinoyl)guanidino]pentaneDihydrochloride (Ie •2 HCl)

[0227] The combined mother liquors from the crystallization of Ie weretreated with powder KOH until the solution reached pH=11. Theprecipitate that formed was collected by vacuum filtration, washed withwater, and recrystallized twice from 10% aqueous HCL to give Ie •2HCl asa pale yellow solid: 27.3 mg (13% yield from III); ¹H NMR (360 MHz,DMSO-d₆) δ1.40 (m, 2H), 1.61 (m, 4H), 3.34 (m, 4H), 7.41 (br s, 4H),8.80 (br s, 2H), 8.93 (br S, 2H), 9.29 (s, 2H) and 10.52 (s, 2H); ¹³CNMR (90 MHz DMSO-d₆) δ23.1, 27.3, 40.8, 109.0, 119.7, 153.2, 154.2,155.9 and 165.2; API MS m/z=527 [C₁₇H₂₄Cl₂N₁₄O₂+H]⁺; HPLC (Method E)95.2%, t_(r)=5.78 min.

EXAMPLE 9

[0228] 1,4-Bis[(35-diamino-6-chloropyrazinoyl)guanidino]xylyleneDihydrobromide (If)

[0229] Compound If was prepared following the same procedure describedfor Ib. The Cbz-protected pseudothiourea III (280 mg, 0.7 mmol) and1,4-xylylenediarnine (IVf, 50 mg, 0.30 mmol) were reacted in thepresence of triethylamine (480 μL, 3.4 mmol) and mercury(II) chloride(192 mg, 0.7 mmol). The reaction mixture was stirred at rt for 4 d andthen it was filtered through silica gel and concentrated under reducedpressure. The residue was suspended in anhydrous DMF (10 mL) and treatedwith tetrabutylammonium borohydride (50 mg, 0.17 mmol) in DMF (1 mL) andstirred for 15 min at rt to get rid of residual mercury(II) chloride.The reaction mixture was filtered thlough silica gel and concentrated togive a residue (Vf) that was treated with 30% HBr in acetic acid (20 mL)for 7 d at rt and 1 d at 45° C. The reaction mixture was poured intoether (200 mL) and the solid that precipitated was collected byfiltration, washed with ether, THF and crystallized twice from methanolto give a pale yellow solid: 74 mg (31% yield from III); ¹H NMR (360MHz, DMSO-d₆) δ4.60 (d, J=4.3 Hz, 4H), 7.45 (s, 4H), 8.91 (br s, 2H),8.99 (br s, 2H), 9.60 (s, 2H) and 10.56 (s, 2H); ¹³C NMR (90 MHz,DMSO-d₆) 6 44.1, 109.1, 119.7, 128.1, 135.4, 153.3, 154.3, 156.0 and165.3; API MS m/z=561 [C₂₀H₂₂Cl₂N₁₄O₂+H]⁺; HPLC (Method E) 95.7%,t_(r)=6.31 min.

EXAMPLE 10

[0230]1,8-Bis[(3,5-diamino-6-chloropyrazinoyl)guanidino]-3,6-dioxa-octaneDihydrochloride (Iv)

[0231] A solution of 2,2′-(ethylenedioxy)bis(ethylamine) (IVg, 45 mg,0.3 mmol) in dry DMF (100 μL was added to III (240 mg, 0.7 mmol) in dryDMF (30 mL), followed by addition of triethylamine (480 μL, 3.4 mmol) inDMF (1 mL) and mercury(II) chloride (165 mg, 0.6 mmol) in DMF (600 μL).The reaction mixture was stirred 16 h at room temperature, thenadditional III (20 mg) was added and reaction mixture was stirred anadditional 8 h at 40° C. The reaction mixture was cooled to roomtemperature and treated with tetrabutylammonitun borohydride (50 mg,0.17 mmol) in DMF (1 mL) with stirring for 15 min at room temperature.The reaction mixture was filtered through silica gel, concentratedLinder reduced pressure to give a solid residue. This was dissolved in30% HBr in acetic acid (20 mL) and stirred for 8 h at 40° C., thenpoured into ether (200 mL). The resulting precipitate was collected byfiltration and washed with ether. The solid was dissolved in water (25mL), the solution filtered, and the filtrate concentrated under reducedpressure. The resulting residue was dissolved again in minimal 10% HBrand powdered NaOH is added to pH=11. A precipitate formed and wascollected by filtration, washed with water and dried to give the freebase (98 mg, 56% yield). A portion of this material (58 mg) wasdissolved in 10% HCl and then concentrated under reduced pressure. Theresidue is washed with ether and dried to give Ig as a pale yellowsolid: 64 mg (32% yield from III); ¹HNMR (360 MHz, DMSO-d₆) δ3.56 (br s,4H), 3.63 (br s, 8H), 7.4 (br s, 4H), 9.09 (br s, 4H), 9.52 (br s, 2H)and 10.70 (s, 2H); ¹³C NMR (90 MHz, DMSO-d₆) δ41.1, 67.7, 69.5, 108.9,119.7, 153.3, 154,2, 155.8 and 165.3; API MS m/z =573[C₁₈H₂₆Cl₂N₁₄O₄+H]⁺; LC (Method C) 97.6%, t_(r)=4.23.

EXAMPLE 11 Potency of Dimeric Compounds

[0232] Two pharmacologic assays were used to determine the relativepotency of the dimers described herein. The first assay examined theexpression of the subunits α, β, and γ of recombinant apical membraneepithelial Na⁺ channel (or “rENaC”) in Xenopus oocyte, as follows: cRNAsfor all three ENaC subunits were injected into oocytes via conventionalmicroinjection techniques. After two to three days, two electrodevoltage clamp protocols were used to measure ENaC-mediated Na⁺ currents.Test compounds were assayed using cumulative drug addition-protocolsknown in the art. Single oocytes were used for single compounds.Compounds tested were then compared to dose-effect relationships foramiloride and benzamil in the same batch of injected eggs.

[0233] In the second potency assay, airway epithelial monolayers mountedwere in Ussing chambers: The principal assay consisted of tests oflumenal drug inhibition of airway epithelial Na+currents. Cells obtainedfrom freshly excised human or dog airways were seeded onto SNAP-wellInserts (CoStar), cultured under air-liquid (ALI) conditions inhormonally defined media. The cells were assayed for Na⁺ transportactivity while bathed in Krebs bicarbonate Ringer (KBR) in the Ussingchambers under voltage clamp conditions. All test drug additions were tothe mucosal bath with half-log dose addition protocols (10⁻¹¹M -10⁻⁵ M).All drugs were made in standard stocks of 10⁻² M drug in DMSO. Eightpreparations were typically run in parallel; two preparations/run wereroutinely used to assay amiloride and benzamil. After the maximalconcentration (10⁻⁴ M) was administered, the lumenal bath was exchangedthree times with fresh KBR solution, which was defined as the “wash-out”effect. All data from the voltage clamps were collected via a computerinterface and analyzed off-line.

[0234] Dose-effect relationships for all compounds were considered andanalyzed by the Prism 3.0 program. The IC₅₀, maximal effectiveconcentrations, and percent washout were calculated and compared tothose of amiloride and benzamil as reference compounds.

EXAMPLE 13 Absorption Assays

[0235] Compounds useful in the present invention preferably have thecharacteristics of high potency and non- or decreased absorbability intomucosal surfaces. Two pharmacologic assays were employed to test theabsorption of compounds illustrated in TABLE 1.

[0236] The first assay is referred to an assay of reversibility.Empirically, the percent of wash-out correlates with cellular uptake.The relationship is complex because reversibility is also a function ofpotency. However, reversibility is a quick and simple screen. An exampleof the information obtained by such as assay is shown in FIG. 1. Thecompounds that “reverse” best in this assay referenced to benzamil werealso the least absorbed in the confocal assay, as described below.

[0237] The second assay utilizes confocal microscopy of amiloridecongener uptake: Virtually all amiloride-like molecules fluoresce in UVrange. This property of these molecules was used to directly measurecellular uptake, using a x-z confocal microscope (Leica). As an exampleof the results obtained by this assay are shown in FIG. 2. In theexperiment shown in FIG. 2, equimolar concentrations of amniloride andcompounds of rapid (benzamil) and very rapid uptake (phenamil) wereplaced on the apical surface of airway cultures on the stage of theconfocal microscope. Serial x-z images were obtained with time and themagnitude of fluorescence accumulating in the cell compartmentquantitated and plotted. The assay was subsequently optimized to testfor compounds that absorbed into cells less rapidly than amiloride. Twocompounds from the synthesis series described above (CF-509 and CF-519)appear to fulfil this criterion. Compounds that were equipotent orgreater with amiloride were tested for wash-out as described above.However, because wash-out may reflect both potency and cell uptake, therate of accumulation of fluorescence (indexed to the specificfluorescence/emission spectrum of each compound) in the cell compartmentas a function of time was also routinely measured. The relative cellularuptake of each test compound was then compared to the referencecompounds (amiloride, benzamil) as for potency assays.

[0238] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defm,ed by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A compound of the formula P₁-L-P₂, wherein:P₁ is a pyrazinoylguanidine sodium channel blocker; L is a linkinggroup; and P₂ is selected from the group consisting ofpyrazinoylguanidine sodium channel blockers and P2Y₂ receptor agonists;or a pharmaceutically acceptable salt thereof.
 2. A compound accordingto claim 1, wherein P₂ is a pyrazinoylguanidine sodium channel blocker.3. A compound according to claim 2, wherein: P₁ and P₂ are eachindependently selected from the group consisting of compounds of theformula:

wherein: X is selected from the group consisting of chloro, bromo, iodo,loweralkyl, lower-cycloalkyl having from-3 to 7 carbons, phenyl,chlorophenyl, bromophenyl, Z-thio and Z-sulfonyl wherein Z is selectedfrom the group consisting of loweralkyl, oxyalkyl, andphenyl-loweralkyl.; Y is selected from the group consisting of hydroxyl,mercapto, loweralkyloxy, loweralkylthio, chloro, loweralkyl,lowercycloalkyl having from 3 to 6 carbons, phenyl, and amino having thestructure:

wherein: R is selected from the group consisting of hydrogen, amino,amidino, lower-cycloalkyl having 3 to 6 carbon atoms, loweralkyl,hydroxyloweralkyl, halo-loweralkyl, lower-(cycloalkylalkyl) having 3 to6 carbons in the ring, phenyl-loweralkyl, lower-(alkylaminoalkyl),lower-alkenyl, phenyl, halophenyl, and lower-alkylphenyl; R₁ is selectedfrom the group consisting of hydrogen, loweralkyl, loweralkenyl, andadditionally; R and R₁ can be joined to form a lower alkylene; R₂ isselected from the group consisting of hydrogen and loweralkyl; R₃ and R₄are independently selected from the group consisting of hydrogen,loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1 -pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl; and L is selected from thegroup consisting of loweralkyl, hydroxy-loweralkyl, phenyl-loweralkyl,(halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-l-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl.
 4. A compound according toclaim 1, wherein said P₂ is a nucleotide or dinucleotide P2Y₂ receptoragonist.
 5. A compound according to claim 4, wherein P₂ is adinucleotide having the formula:

wherein: A and B are each independently a purine or pyrimidine base; R₁and R₂ are each independently selected from the group consisting of H orOH; and n is from 1 to 6; with said dinucleotide covalently joined to Lby covalent attachment to A or B or covalent attachment to the ribose ordeoxyribose ring to which A and B are joined.
 6. A compound of claim 1,wherein the compound has the structure:


7. A compound of claim 1, wherein the compound has the structure:


8. A compound of claim 1, wherein P₁ is selected from the groupconsisting of amiloride, benzamil and phenamidl.
 9. A pharmaceuticalformulation comprising a compound according to claim 1 in apharmaceutically acceptable carrier solution.
 10. A method of treating amucosal surface in a subject in need thereof, comprising administeringto said subject a compound according to claim 1 in an amount effectiveto treat said mucosal surface.
 11. A method according to claim 10,wherein said mucosal surface is an airway mucosal surface.
 12. A methodaccording to claim 10, wherein said mucosal surface is agastrointestinal mucosal surface.
 13. A method of treating a mucosalsurface in a subject in need thereof, comprising administering to saidsubject, in an amount effective to treat a mucosal surface, a compoundcomprising a covalent conjugate of a pyrazinoylguanidine sodium channelblocker and a non-absorbable carrier moiety.
 14. A method according toclaim 13, subject to the proviso that said mucosal surface is not anasal mucosal surface.
 15. A method according to claim 13, wherein saidmucosal surface is an airway mucosal surface.
 16. A method according toclaim 13, wherein said mucosal surface is a gastrointestinal mucosalsurface
 17. A method according to claim 13, wherein said conjugate hasthe formula: formula:

wherein: X is selected from the group consisting of chloro, bromo, iodo,loweralkyl, lower-cycloalkyl having from 3 to 7 carbons, phenyl,chlorophenyl, bromophenyl, Z′-thio and Z′-sulfonyl wherein Z′ isselected from the group consisting of loweralkyl and phenyl-loweralkyl.;Y is selected from the group consisting of hydroxyl, mercapto,loweralkyloxy, loweralkylthio, chloro, loweralkyl. lowercycloalkylhaving from 3 to 6 carbons, phenyl, amino having the structure:

wherein: R is selected from the group consisting of hydrogen, amino,amidino, lower-cycloalkyl having 3 to 6 carbon atoms, loweralkyl,hydroxyloweralkyl, halo-loweralkyl, lower-(cycloalkylalkyl) having 3 to6 carbons in the ring, phenyl-loweralkyl, lower-(alkylaminoalkyl),lower-alkenyl, phenyl, halophenyl, and lower-alkylphenyl; R₁ is selectedfrom the group consisting of hydrogen, loweralkyl, loweralkenyl, andadditionally; R and R₁ can be joined to form a lower alkylene; R₂ isselected from the group consisting of hydrogen and loweralkyl; R₃ isselected from the group consisting of hydrogen, loweralkyl,hydroxy-loweralkyl, phenyl-loweralkyl, (halophenyl)-loweralkyl,lower-(alkylphenylalkyl), (loweralkoxyphenyl)-loweralkyl,naphthyl-loweralkyl, (octahydro-1-azocinyl)-loweralkyl,pyridyl-loweralkyl, and loweralkyl radicals linked to produce with thenitrogen atom to which they are attached a 1-pyrrolidinyl, piperidino,morpholino, and a 4-loweralkyl-piperazinyl group, and phenyl; and L isselected from the group consisting of loweralkyl, hydroxy-loweralkyl,phenyl-loweralkyl, (halophenyl)-loweralkyl, lower-(alkylphenylalkyl),(loweralkoxyphenyl)-loweralkyl, naphthyl-loweralkyl,(octahydro-1-azocinyl)-loweralkyl, pyridyl-loweralkyl, and loweralkylradicals linked to produce with the nitrogen atom to which they areattached a 1-pyrrolidinyl, piperidino, morpholino, and a4-loweralkyl-piperazinyl group, and phenyl; and Z is a non-absorbablecarrier moiety covalently linked to the adjacent nitrogen atom; or apharmaceutically acceptable salt thereof.